Cloning and characterization of acetylcholinestera 1genes from incticide-resistant field populations of Liposcelis paeta Pearman (Psocoptera:Liposcelididae)
Shuang Wu a ,b ,Ming Li a ,Pei-An Tang a ,c ,Gary W.Felton b ,Jin-Jun Wang a ,*
a
Key Laboratory of Entomology and Pest Control Engineering,College of Plant Protection,Southwest University,Chongqing 400715,PR China b
Department of Entomology,Center for Chemical Ecology,Penn State University,University Park,PA 16802,USA c
School of Food Science and Engineering,Nanjing University of Finance and Economics,Nanjing 210003,PR China
a r t i c l e i n f o
Article history:
石壕村
Received 11February 2010Received in revid form 23March 2010
Accepted 1April 2010
Keywords:
Liposcelis paeta Pearman ace gene
Molecular cloning
Comparative quantitative expression Incticide resistance
a b s t r a c t
The psocid,Liposcelis paeta Pearman,is an increasingly important polyphagous pest of stored products worldwide.Intensive u of organophosphorous incticides for pest control has facilitated resistance development in psocids in China.Three incticide-resistant field populations of L .paeta were collected from Nanyang city of Henan Province (NY),and Wuzhou (WZ)and Hezhou (HZ)cities of Guangxi Province,China.Previous studies have shown that psocids have different susceptibilities to incticides.In addition,their AChE susceptibilities to paraoxon-ethyl and demeton-S-methyl also differed from each other.Acetylcholinestera 1,which is one of the major targets for organophosphate incticides,has been fully cloned and quenced from the populations of L .pae
ta .Comparison of both nucleotide and deduced amino acid quences revealed nucleotide polymorphisms among L .paeta ace 1genes from different populations,but none of the polymorphisms correspond to the active sites in AChE 1from other incts.The results of comparative quantitative real-time PCR indicated that the relative expression level of HZ ace 1gene was the highest among three populations,which was 1.20and 1.02-fold higher than tho of NY and WZ populations,respectively.This may due to an epigenetic inheritance phenomenon,which allows organisms to respond to a particular environment through changes in gene expression.
Ó2010Elvier Ltd.All rights rerved.
1.Introduction
Acetylcholinestera (AChE;EC 3.1.1.7)encoded by the ace gene is a key enzyme in the nervous system existing on the cholinergic post-synaptic membrane which plays a main role in the termina-tion of signal transmission by hydrolyzing excess amounts of acetylcholine into acetic acid and choline in the synaps and neuromuscular junctions (Oakeshott et al.,2005).AChE is the major target for organophosphate and carbamate incticides,which inhibit enzyme activity by covalently phosphorylating or carba-mylating the rine residue within the active site gorge (Corbett,1974).Sev
eral studies have reported that both quantitative and qualitative changes in AChE conferred resistance to incticides (Fournier et al.,1992,1993).In incts,two different ace genes have been found in various species,such as Anopheles gambiae (Weill et al.,2002),Aphis gossypii (Li and Han,2002),Culex pipiens
(Huchard et al.,2006),Aedes aegypti (Mori et al.,2007),Myzus persicae (Nabeshima et al.,2003)and Bombyx mori (Seino et al.,2007).Some studies indicated that ace 1gene transcripts are much more abundant than ace 2.The majority of the functions of AChEs,including synaptic neurotransmission have been ascribed to ace 1(Alout et al.,2007;Lee et al.,2007).
Liposcelis paeta Pearman (Psocoptera:Liposcelididae)is an important polyphagous pest of stored food products and commonly found in flours,cereals and rice (Turner,1994).Infestations have also been found in herbaria (Rethief et al.,1995),papers,books,and inct collections (Turner,1987).Like other species of psocids,L .paeta is a potential cau of respiratory and dermatological allergies (Turner et al.,1996;Müsken et al.,1998;Patil et al.,2001).Outbreaks of psocids have been reported in many countries such as Australia,Indonesia,Thailand,and the People ’s Republic of China (Wang and Zhao,2003;Leong and Ho,1995).In Australia,detection of a high level resistance to phosphine in psocids infesting stored commodities has elevated their pest status enormously (Nayak et al.,2003).
Moreover,the importance of psocids in Australia has been enhanced in recent years,mostly due to the failure of almost
*Corresponding author.Tel.:þ862368250255;fax:þ862368251269.E-mail address: (J.-J.
Wang).Contents lists available at ScienceDirect
Inct Biochemistry and Molecular Biology
journal ho mep age:www. m/lo
cate/ibmb
0965-1748/$e e front matter Ó2010Elvier Ltd.All rights rerved.doi:10.1016/j.ibmb.2010.04.001
Inct Biochemistry and Molecular Biology 40(2010)415e 424
all currently registered grain protectants against the pests (Nayak et al.,2005;Nayak and Daglish,2006,2007).In China, intensive u of organophosphorous incticides in pest control also facilitated resistance development in psocids.Especially in Henan and Guangxi Province,vere infestation caud by psocids has been reported frequently in recent years,and L.paeta is one of the most widespread important species in the areas.
Bad on our previous studies,which have provided basic information about detoxifying and target enzymes in the psocid pest genus Liposcelis(Dou et al.,2006;Chai et al.,2007;Cheng et al., 2007;Ren et al.,2008;Wu et al.,2009),the molecular cloning and characterization of acetylcholinester
a1genes(ace1)from incticide-resistantfield populations of L.paeta were investigated in this study.This helps elucidate the molecular characterization of an incticide-resistant associated target enzyme in L.paeta and provides information crucial to future pest management.
2.Materials and methods
2.1.Incts
Nymphs of L.paeta were collected from wheat warehous in Nanyang(NY population)of Henan Province,Wuzhou(WZ pop-ulation)and Hezhou(HZ population)of Guangxi Province,China in 2004.Incts were reared on a diet consisting of whole-wheatflour, skimmed milk,and yeast powder(10:1:1)in an air-conditioned room at27Æ0.5 C,75e80%RH and a scotoperiod of24h.All experiments were conducted under the conditions described above with three to five day-old adult females.The toxicities(LC50)of dichlorvos against the three populations were281.5,285.1,and243.5mg/m2for NY, WZ,and HZ populations,respectively(Ren et al.,2008).In addition, the toxicities of chlorpyrifos,cypermethrin,and carbosulfan against the three populations also varied signifi,the highest LC50 value of chlorpyrifos(34.9mg/m2)for HZ population,but the lowest (16.8mg/m2)for NY population;the highest LC50value of cyper-methrin(33.7m g/m2)for N
Y population,but the lowest(7.3m g/m2) for WZ population;the highest LC50value of carbosulfan(7.4mg/m2) for HZ population,but the lowest(5.8mg/m2)for WZ population] (P<0.05).The bioassay was carried out at27Æ0.5 C for30min (Ren et al.,2008;Wu et al.,2009).
2.2.RNA extraction and cDNA synthesis
Total RNA was extracted from threefield populations of L.paeta female adults(200mg)using Trizol reagent(Invitrogen,Carlsbad, CA,USA).In all samples,total RNA was treated with DNa I(Takara, Kyoto,Japan).Total RNA concentration was determined by measuring ultraviolet absorbance at260nm.RNA purity was checked by determining the A260/A280ratio and its integrity was checked by formaldehyde agaro gel electrophoresis.First-strand cDNA was immediately synthesized after extraction using a cDNA synthesis kit(Takara).To amplify the partial cDNA fragment of ace1 gene,degenerate primers(n primer:50-GCNACNATGTGGAAYC CNAA-30,anti-n primer:50-RTTYTTCCARAANGCRCAYTG-30) were designed by alignment of AChE amino acid quences derived from other inct species.Polymera Chain Reaction(PCR)was carried out with a TGradient PCR Thermal Cycler(Biometra)in a50m L reaction mixture containing0.2m g of cDNA as a template in PCR buffer containing 3.5mM MgCl2,0.2mM dNTPs(deoxy-nucleotide triphosphates),0.4m M of each primer and two units of Taq polymera(Takara).After3min pre-denaturation at94 C,35 amplific
ation cycles were performed as follows:30s at94 C,30s at53 C and1min at72 C,the last extension step was extended to 10min at72 C,followed by cooling to4 C.For the full-length ace1gene,the reaction condition was the same,with the exception that the annealing temperature was changed.
To obtain full-length cDNAs,gene-specific primers(GSPs)for rapid amplification of cDNA ends(RACE)were designed bad on the quences of three cloned ace1cDNA fragments from three populations of L.paeta and using primer premier5.00for Windows ()recommended by the30-Full RACE Core Set Ver.2.0(Takara)for30RACE and BD SMARTÔcDNA Amplification Kit(Clontech)for50RACE.They were as follows:30-GSP for ace1,50-AGTCGGAATTTGTTCAGTCAAGC-30(n primer), 50-TCAGTTCTGGTGGATAACGAGTG-30(nested n primer);50-GSP for ace1,50-TAGTAGCCTTCTTCGGTGTTGGA-30(anti-n primer), 50-TATGACGGGTACGAAAGGAAACT-30(nested anti-n primer). The primers for amplifying the full-length of ace1gene were n primer,50-AAGCAGTGGTATCAACGCAGAGT-30;anti-n primer, 50-CCTTCTTCATTGAAGAATCGTA-30.The PCR products(25m L)were parated by1.0%agaro gel electrophoresis and stained with ethidium bromide(EB).
2.3.Cloning and quencing
羼水的拼音The band of the expected size was excid and the fragment was purified using the Gel Extraction Mini Kit(Watson Biotechnologies, Inc),and then cloned into pGEMÒ-T easy vector(Promega Corp.).The ligation reactions were ud for transformations with Escherichia coli DH5a-competent cells.Successful clones were confirmed by standard ampicillin lection and PCR with primers ud before.Recombinant plasmids were fully quenced in both directions with an ABI Model 3100automated quencer(Invitrogen Life Technologies).
2.4.Sequence analysis and phylogenetic tree construction
Blast rearch was performed bi.v/ blast.The signal peptide was predicted by SignalP 3.0rver program(www.cbs.dtu.dk/rvices/SignalP)(Bendtn et al., 2004).Multiple quence alignment was performed with DNAMAN (DNAMAN 5.2.2,Lynnon BioSoft).ExPASy Proteomics Server (cn.expasy/tools/pi_tool.html)was ud to compute isoelectric point and molecular weight of deduced protein quences.The phylogenetic tree was constructed by the programs of MEGA4.1(Tamura et al.,2007)and clustalx1.81(Jeannmougin et al.,1998)using the neighbor-joining method.Bootstrap values were calculated on1000replications.Hydropathy profile was drawn using protein hydrophobicity plots rver(www.vivo. colostate.edu/molkit/hydropathy/index.html).The AChE quences of different
species were retrieved from GenBank(www. ncbi.v/Genbank/index.html).Homology modeling was completed pasy/swissmod/SWISS-MODEL. html and the structures were viewed with SwissPDBviewer (pasy.ch/spdbv)programs and PyMOL0.99software (DeLano Scientific LLC).Bad on the amino acid identity with quences of known three-dimensional structures,human butyr-ylcholinestera structure(PDB:2J4CA)was ud by Internet modeling rver as the main template to L.paeta AChE1.
2.5.Quantitative real-time PCR
The mRNA levels of ace1from threefield populations of L.paeta were examined utilizing theΔΔCT method(Livak and Schmittgen, 2001)on a Stratagene Mx3000P thermal cycler(Stratagene)with the houkeeping gene b-actin from L.paeta as a reference.Gene-specific primers were designed using Primer3.0(frodo.wi. mit.edu).The efficiency of PCR amplification for gene-specific primers was analyzed by one cDNA sample withfive rial dilutions,three technical replications.The real-time PCR was
S.Wu et al./Inct Biochemistry and Molecular Biology40(2010)415e424 416
S.Wu et al./Inct Biochemistry and Molecular Biology40(2010)415e424417
carried out in 20m L reactions containing 2m L of template cDNA or the standard,10m L Brilliant ÒSYBR ÒGreen QPCR Master Mix (Stratagene),and 0.2m M of each primer:for b -actin,50-ACGAACTCCGAGTCGCCCCA-30(n primer),50-ACAAGGACAACA CGGCCTGG-30(anti-n primer);for ace 1,50-AAAGCAGCTGT AATGGTGTG-30(n primer),50-ATCATGAACCCACTGCAAAG-30(anti-n primer).Thermal cycling conditions were:95 C for 10min,40cycles of 95 C for 30s,60 C for 30s,and 72 C for 30s.After the cycling protocol,a melting curve analysis from 60 C to 95 C was applied to all reactions to ensure consistency and spec-i ficity of the ampli fied product.Data were statistically analyzed using analysis of variance (ANOVA),and the means were parated by Duncan ’s Multiple Range Test for signi ficance (P ¼0.05)by using SPSS 12.0for Windows (SPSS,Inc.,Chicago,IL,USA).3.Results
水葫芦的功效与作用3.1.Sequence analysis
The complete ace 1cDNA genes from three field populations of L .paeta all consist of 2509bp with an open reading frame (ORF)of
2358nucleotides,encoding a protein of 785amino acids residues (Fig.1A).They all include a 50untranslated region (UTR)located 125bp upstream of the putative start codon (ATG)and the stop c
odon (TGA)located at position 2481e 2483.No polyadenylation signal (AATAAA)was found within the 30-UTR,although a 20bp span of poly (A)nucleotides was detected at its end.Bad on the deduced amino acid quences,the theoretical molecular weights of ace 1from NY,HZ and WZ populations were calculated to be 89.48,89.31and 89.34kDa,with isoelectric points (pI)of 6.25,6.33and 6.33,respectively.
The quences have been deposited in GenBank (accession numbers:GU214754e GU214756).Sequence BLAST showed that the deduced amino acid quences of ace 1from NY,HZ and WZ pop-ulations of L .paeta were highly conrved with the AChE 1homolog in other psocid species,sharing 94.50,93.86and 93.99%identity with Liposcelis bostrychophila AChE 1;93.33,92.70and 92.70%identity with Liposcelis entomophila AChE 1;91.40,90.80and 90.80%identity with Liposcelis decolor AChE 1,respectively.Phylo-genetic tree (Fig.2)also demonstrates that they belong to inct AChE 1,and shows their relationships with different AChEs from other
species.
Fig.1.(continued ).
Fig.1.The nucleotide and deduced amino acid quence alignment of ace 1among three field populations of L .paeta (GenBank accession numbers:GU214754e GU214756).A.The 50and 30UTRs are lowercad;arrows indicate the position of degenerate primers,and speci fic primers ud in the 30and 50ends ampli fication are underlined.The start and stop codon are highlighted in bold italic.B.AChE from T.californica (CAA27169)was ud to compare the amino acid quence differences.The connecting lines symbolize the cysteines forming the disul fide bonds.The solid triangles indicate the residues involved in the catalytic triad.The hollow pentacles show the oxyanion hole and the solid pentacle indicates the choline-binding site.The pound keys reprent the conrved aromatic amino acid residues.The hollow triangles show the deduced amino acid differences among three populations of L.paeta .
S.Wu et al./Inct Biochemistry and Molecular Biology 40(2010)415e 424
418
The deduced amino acid quences of ace 1exhibit all the common features of AChE,including conrved active site triad,S405,E531and H645;a choline-binding site W290;3pairs of cysteine residues forming intra-chain disul fide bonds (C273e 300,C459e 472,C607-729);oxyanion hole-forming residues G324,G325,A406;other 8conrved aromatic amino acid residues,including one of the acyl pocket-forming residue F494;the quence FGESAG conrved in all cholinesteras (Fig.1B).Furthermore,bad on the structure and quence of Torpedo cal-ifornica AChE (CAA27169)and the quences of L .bostrychophila (ACN78619),L .entomophila (ACI16651)and Drosophila mela-nogaster (AAF54915),other characteristic residues of AChE were found in L.paeta AChE 1(Table 1)(Sussman et al.,1991).However,some of them were not so conrved among different species,including the other two residues forming the choline-binding site:Y534,S503in L .paeta (F351,E320in T .californica ,Y408,A377in D .melanogaster );another residue composing the acyl pocket:C492in L .paeta (F309in T .californica ,L366in D .melanogaster );one residue forming the peripheral anionic site:I276in L .paeta (Y91in T .californica ,E107in D .melanogaster );residues composing the internal surface of active site gorge:I276,D647,C492,Y534in L .paeta (Y91,Y463,F309,F351in T .californica ,E107,D520,L366,Y408in D .melanogaster ).Except for tho residue differences,other deduced amino acids are still the same.
According to nucleotide quence alignment among three populations of L .paeta ,there are totally 5out of 7differences between the full-length ace 1cDNA of L .paeta from Guangxi and Henan Province (Fig.1A),resulting in 5deduced amino acid differences among three populations,and 4of them exist between L .paeta from two Provinces (Fig.1B).3.2.Putative protein characters
Despite no signal peptide was found within the L .paeta AChE 1precursors,which are the same as Blattella germanica (ABB89946)and L .entomophila (ACI16651),hydropathy pro files of AChE 1from three field populations of L .paeta are quite similar to AChEs from B .germanica (ABB89946),L .entomophila (ACI16651)and L .bos-trychophila (ACN78619),D .melanogaster (AAF54915)with signal peptides cutoff (Fig.3).Like other AChEs,the hydrophobic region in the C-terminus of L .paeta AChE 1,clo to I773,demonstrates the prence of a glycolipid anchor (Haas et al.,1988;Kozaki et al.,2008).
Protein modeling bad on the structural model of human butyrylcholinestera (PDB:2J4CA)indicated that three-dimen-sional structures of NY,HZ and WZ L .paeta AChE 1are folded similarly to human butyrylcholinestera (Fig.4A),although they only show 46.44,46.26and 46.26%quence identity with
it,
Liposcelis paeta HZ Liposcelis paeta WZ Liposcelis paeta NY
Liposcelis entomophila -1 Liposcelis bostrychophila -1 Liposcelis decolor -1 Blattella germanica -1 Bombyx mori
Bombyx mandarina Helicoverpa armigera Helicoverpa assulta Bemisia tabaci Aphis gossypii
Rhopalosiphum padi Sitobion avenae -1 Lutzomyia longipalpis Anopheles gambiae Aedes albopictus
Culex tritaeniorhynchus
Caenorhabditis elegans -1
Boophilus microplus -1
Torpedo californica Gallus gallus
Bos taurus
Homo sapiens
Mus musculus Rattus norvegicus
Drosophila melanogaster Lucilia cuprina Aedes aegypti
Sitobion avenae -2胡萝卜丸子的做法
Blattella germanica -2
Nilaparvata lugens
Liposcelis decolor -2
Liposcelis bostrychophila -2 Liposcelis entomophila -2
Caenorhabditis elegans -2 Caenorhabditis elegans -3 Boophilus microplus -3
Boophilus microplus -2
100
元日战争100100
100
10097
100
100
6798
72
100
99100
镇魂经典语录
96
50
79
786363
100
64
99读书有益
100
100
100100
100
89100
9047781009181
87Inct AChE1 Nematode AChE1 Tick AChE1 Vertebrate AChE1 Inct AChE2 Nematode AChE3-4
Tick AChE2-3 Fig.2.The molecular evolutionary tree of acetylcholinestera with 40amino acid quences of acetylcholinestera from 29species.The inct AChEs fall into two different怎样让全身变白
subgroups.The corresponding GeneBank accession numbers phila-1,ACI16651;L.bostrychophila-1,ACN78619;L.decolor-1,anica-1,i ,ABY50088;B.mandarina ,ACL80033;H.armigera ,AAY59530;H.assulta ,AAY42136;B.tabaci ,ssypii ,AAM94376;R.padi ,AAT76530;S.avenae-1,AAV68493;L.longipalpis ,ABI74669;A.gambiae ,CAD56157;A.albopictus ,itaeniorhynchus ,BAD06210;C.elegans-1,NP_510660;B.microplus-1,CAA11702;T.californica ,CAA27169;G.gallus ,NP_990749;B.taurus ,AAI23899;H.sapiens ,AAA68151;M.musculus ,vegicus ,NP_lanogaster ,AAF54915;L.cuprina ,AAC02779;A.aegypti ,AAB35001;S.avenae-2,anica-2,ABB89947;N.lugens ,CAH65679;L.decolor-2,FJ647187;L.bostrychophila-2,phila-2,EU854150;C.elegans-2,NP_496963;C.elegans-3,AAC14017;B.microplus-3,AAP92139;B.microplus-2,CAB93511.
S.Wu et al./Inct Biochemistry and Molecular Biology 40(2010)415e 424419
